A single region of the Phytophthora infestans avirulence effector Avr3b functions in both cell death induction and plant immunity suppression

Abstract As a destructive plant pathogen, Phytophthora infestans secretes diverse host‐entering RxLR effectors to facilitate infection. One critical RxLR effector, PiAvr3b, not only induces effector‐triggered immunity (ETI), which is associated with the potato resistance protein StR3b, but also suppresses pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). To date, the molecular basis underlying such dual activities remains unknown. Based on phylogenetic analysis of global P. infestans isolates, we found two PiAvr3b isoforms that differ by three amino acids. Despite this sequence variation, the two isoforms retain the same properties in activating the StR3b‐mediated hypersensitive response (HR) and inhibiting necrosis induced by three PAMPs (PiNpp, PiINF1, and PsXeg1) and an RxLR effector (Pi10232). Using a combined mutagenesis approach, we found that the dual activities of PiAvr3b were tightly linked and determined by 88 amino acids at the C‐terminus. We further determined that either the W60 or the E134 residue of PiAvr3b was essential for triggering StR3b‐associated HR and inhibiting PiNpp‐ and Pi10232‐associated necrosis, while the S99 residue partially contributed to PTI suppression. Additionally, nuclear localization of PiAvr3b was required to stimulate HR and suppress PTI, but not to inhibit Pi10232‐associated cell death. Our study revealed that PiAvr3b suppresses the plant immune response at different subcellular locations and provides an example in which a single amino acid of an RxLR effector links ETI induction and cell death suppression.


| INTRODUC TI ON
The oomycete pathogen Phytophthora infestans causes late blight diseases and has threatened global food production since the 19th century Irish Potato Famine. As the most destructive pathogen of potatoes, P. infestans is infamous for its rapid spread and frequent resistance (Fry, 2008;Fry et al., 2015). P. infestans manipulates the host immune system by orchestrating an arsenal of cell-entering RxLR effectors. Some RxLR effectors are avirulence (Avr) proteins that exhibit specific gene-for-gene interactions with their cognate host resistance (R) proteins, leading to effector-triggered immunity (ETI) and often eliciting a hypersensitive response (HR) (Naveed et al., 2020). Most characterized R proteins in the Phytophthoraplant pathosystem are nucleotide-binding, leucine-rich-repeat receptors (NLRs) that indirectly interact with corresponding Avr proteins. For example, interactions between PiAvr2 and R2 and between PiAvrvnt1 and Rpivnt1 are indirect and mediated by BSL1 and GLYK, respectively (Gao et al., 2020;Saunders et al., 2012).
Sequence polymorphisms, gene deletion, copy number variation, and transcriptional dynamics of AVR genes result in a spectrum of virulence among Phytophthora isolates (Dong & Ma, 2021). For instance, the residues at positions 80 and 103 of Avr3a vary between avirulent and virulent isolates of P. infestans and determine elicitation of the R protein R3a (Armstrong et al., 2005). Deletion or silencing of PiAVR2 results in loss of avirulence in P. infestans on plants harbouring the R2 gene (Gilroy et al., 2011). In several studies, the dual activities of RxLR effectors related to cell death activation and host immune suppression were linked to different amino acids.
For example, the K80 and I103 residues of P. infestans PiAvr3a KI determined R3a-mediated HR, while the Y147 residue was required for PiINF1 suppression but not R3a activation (Bos et al., 2009).
Mutational analysis of Phytophthora sojae Avh238 indicated that the 79th residue determined cell death-inducing activity, and the 53rd amino acid in its C-terminal region was critical for promoting infection (Yang et al., 2017).
Studies have also shown that subcellular localization of effectors impacts their activity. Transient expression of 52 infection-inducing RxLR effectors in N. benthamiana revealed that most effectors localize in the cytoplasm, nuclei, or associate with the plasma membrane, while only a few localize in other subcellular compartments (Wang et al., 2019). The diversity of localization patterns suggests that subcellular localization plays a critical role in plant defence responses.
On the other hand, R protein recognition also promotes the relocalization of the corresponding effectors. In N. benthamiana, the nuclear-cytosolic RxLR effector PiAvr3a KI moved to endosomes with R3a to initiate HR (Engelhardt et al., 2012). Activating R1-mediated HR required nuclear localization of Avr1, while Avr1-mediated cell death suppression required its localization in the cytoplasm (Du et al., 2015). Similarly, nuclear localization of P. sojae Avh238 was essential for triggering cell death, while cytoplasmic localization of the effector was needed for Avh238-mediated suppression of INF1triggered cell death (Yang et al., 2017). P. infestans PiAvr3b encodes a cytoplasmic RxLR effector and activates multiple R genes that have been extensively employed in the disease-resistance breeding of potatoes (Rietman, 2011).
Nevertheless, PiAvr3b elicits hypersensitive cell death in wild potato (Solanum pinnatisectum), which is highly resistant to late blight disease (Gu et al., 2020), suggesting cognate R genes are valuable for durable resistance. The cloning of StR3b and its matching avirulence gene PiAVR3b (Li et al., 2011) provides a new tool for understanding the coevolution of host and pathogen machinery in potato and P. infestans, respectively. Without a corresponding StR3b protein, PiAvr3b could function as a virulence factor to suppress immune responses induced by the PAMP PiNpp (Zuluaga et al., 2016) and the RxLR effector Pi22798 , suggesting a potential virulence function of this protein. PiAVR3b deletions and single-nucleotide polymorphisms (SNPs) (Rietman, 2011;Thilliez et al., 2019) may indicate adaptive changes occurring in the genomes of P. infestans isolates that may promote host immune surveillance evasion. However, the variation patterns and structural basis underlying the dual activities of PiAvr3b remain largely unknown.
In this study, we examined sequence variation and changes in the transcription of PiAvr3b and analysed the regions and residues responsible for ETI induction and PTI suppression. We also investigated the correlation between subcellular localization of PiAvr3b and its activities in regulating plant defence. Our study revealed that the C-terminal region of PiAvr3b promotes infection by interfering with multiple subcellular processes of plant immunity, and correct subcellular localization is critical for the dual activities of PiAvr3b.
Our findings provide an example in which a single amino acid in an RxLR effector regulates ETI elicitation and PTI suppression.

| SNPs, genomic deletion, and transcriptional variation of PiAVR3b among P. infestans field isolates
To investigate PiAVR3b sequence variation in P. infestans, we PCRamplified the PiAVR3b coding regions from 95 geographically distinct isolates (Table S1). We could not amplify the AVR3b open reading frames (ORFs) from 27 isolates, indicating that the loci may be disrupted in these isolates ( Figure S1). Sanger sequencing of the remaining 68 PCR-amplicons revealed that the amino acid polymorphisms occurred mainly at the 46th, 93rd, and 133rd residues, which classified PiAvr3b into two isoforms: PiAvr3b RGR and PiAvr3b LRK (Figure 1). We further examined the transcription of the AVR3b loci that were amplified and found that AVR3b was silenced in 42 isolates ( Figure S2 and Table S2). Strikingly, we found that the isolates collected recently showed a much higher frequency of PiAVR3b disruption or silencing (Table S2), indicating a dominant lineage alternation in the Chinese P. infestans population.
Further BLAST analysis using the AVR3b ORF as the query sequence revealed a paralogous gene, Pi18221. Compared to PiAVR3b, Pi18221 lacks two nucleotides, which causes a frameshift and is predicted to truncate the protein by 43 amino acids at the C-terminus ( Figure S2a,b). Interestingly, our transcriptional analysis indicated that Pi18221 transcripts were present in both PiAVR3b-silencing and PiAVR3b-expressing isolates ( Figure S3), suggesting that Pi18221 may be a functional effector protein.
To further understand the divergence of PiAvr3b, we conducted homolog searches against other Phytophthora species. PiAvr3b orthologs were identified in several closely related species, including Phytophthora cactorum, Phytophthora idaei, Phytophthora parasitica, Phytophthora betacei, and Phytophthora mirabilis ( Figure 1 and Table S3). The evolution of PiAvr3b correlated with the current speciation model of these Phytophthora species. In addition, we found several PiAvr3b-like effectors in other Phytophthora species, such as Psoj292825 in P. sojae and Ppal37054 in Phytophthora palmivora, through BLAST searches. All of the PiAvr3b-like effectors identified showed low sequence similarity (identity <30%) to PiAvr3b (Figure 1). For these PiAvr3b-like effector proteins, reciprocal homolog searches were unsuccessful, identifying only different effectors, Pi05910 and Pi19831, in P. infestans (Table S3).
Thus, our results indicate that PiAvr3b is only conserved in the Phytophthora Clade I and displays two isoforms and transcriptional variation.

| PiAvr3b orthologues trigger distinct cell deaths indirectly mediated by StR3b
Previous studies demonstrated that PiAvr3b RGR triggers StR3bmediated HR in N. benthamiana and wild potato cultivars (Gu et al., 2020;Li et al., 2011). To examine whether the polymorphisms identified in other PiAvr3b variants affect StR3b-dependent HR in plants, we co-expressed StR3b with mCherry-tagged PiAvr3b variants in N. benthamiana. As shown in Figure 2, both PiAvr3b RGR and PiAvr3b LRK elicited substantial cell death, whereas the truncated PiAvr3b paralogue Pi18221 failed to stimulate StR3b-HR. Most PiAvr3b orthologs from other Phytophthora species showed a necrotic phenotype similar to that of PiAvr3b, except for Ppar05910, Pida370Avr3b, and Pida376Avr3b ( Figure 2a and Table S3).
Interestingly, the two PiAvr3b-like effectors, Psoj292825 (from P. sojae) and Pi19831 (from P. infestans), also triggered HR. N. benthamiana leaves expressing Pi05910, Ppar05910, Pida370Avr3b, or Pida376Avr3b showed significantly less ion leakage than other PiAvr3b orthologs (Figure 2b). Western blot analysis verified expression of each effector protein fusion ( Figure S4), suggesting that the difference in StR3b-dependent HR was not attributable to the instability of PiAvr3b orthologs or PiAvr3b-like proteins.
To test whether PiAvr3b physically binds to StR3b, we conducted a yeast two-hybrid assay and found that neither PiAvr3b RGR nor PiAvr3b LRK bound to StR3b ( Figure S5). Our results indicate that StR3b possesses a broad effector recognition capacity and may interact with effector proteins indirectly.
F I G U R E 1 Sequence analysis of PiAvr3b isoforms and orthologs in selected Phytophthora species. The purple rectangle encompasses RxLR motifs, and the blue rectangles indicate the amino acids that differ between PiAvr3b RGR and PiAvr3b LRK . Phylogenetic analysis of PiAvr3b and its homologs found in Phytophthora. The two PiAvr3b-like effectors Psoj292825 and Ppal37054 from P. sojae and P. palmivora, respectively, were used as outgroups. Numbers in the phylogenetic tree indicate bootstrap values, and the scale bar represents 20% weighted sequence divergence. Numbers in the upper right corner indicate genotype frequencies of PiAvr3b alleles in different P. infestans isolates. Black arrows indicate residues subject to the following mutation analysis.

PiAvr3b-triggered ETI in N. benthamiana
To better understand the signalling pathway modulating PiAvr3b-StR3b recognition, we employed a virus-induced gene silencing (VIGS) strategy (Liu et al., 2002) to silence several critical genes involved in the plant immune response. NbSGT1, NbHSP90, NbMEK2, and NbRAR1 were selected because these genes function as regulators of both PTI and ETI ( Figure S6) (Yang et al., 2017). We found that silencing of NbMEK2 and NbRAR1 did not affect HR triggered by PiAvr3b RGR and StR3b, while silencing of NbHSP90 and NbSGT1 abolished HR ( Figure 3). These results indicate that PiAvr3b-StR3b-triggered cell death requires HSP90 and SGT1 but not NbMEK2 and NbRAR1.

| Both PiAvr3b isoforms promote P. infestans infection by suppressing PAMP-and effector-triggered cell death
As previously mentioned, phylogenetic analyses identified two isoforms of PiAvr3b: PiAvr3b RGR and PiAvr3b LRK . To further delineate their activities and roles in regulating plant defence, both PiAvr3b isoforms were fused to mCherry and transiently expressed in N. benthamiana by agroinfiltration. Compared to the mCherry control, both PiAvr3b RGR and PiAvr3b LRK produced approximately 2-fold larger lesions, which was confirmed by quantitative biomass and sporangial measurements (Figure 4a-d). Pi18221 was not observed to be involved in virulence.
Next, we tested whether the two PiAvr3b isoforms could suppress the burst of reactive oxygen species (ROS) that plants deploy for defence against pathogens. We treated N. benthamiana leaf discs with bacterial flg22, which can induce an ROS burst, and found that neither PiAvr3b RGR nor PiAvr3b LRK suppressed ROS bursts, although the positive control Pi20303 (a paralogous effector of PiAvrblb2; Zheng et al., 2014) exhibited ROS suppression ( Figure 4e). To further determine if either PiAvr3b isoform could inhibit PTI, we expressed three Phytophthora PAMPs (PiNpp, PiINF1, and PsXEG1) in N. benthamiana leaves. We found that both PiAvr3b RGR and PiAvr3b LRK , but not Pi18221, suppressed necrosis induced by all three PAMPs; the positive control PiAvr3a KI  suppressed only PiINF1-and PsXEG1-associated necrosis (Figure 4d,f). We also tested the ability of PiAvr3b RGR and F I G U R E 2 PiAvr3b intra-and interspecies homologs stimulate different StR3b-mediated hypersensitive responses (HR). (a) Co-expression of StR3b and mCherry-tagged PiAvr3b homologs in Nicotiana benthamiana. PiAvr3a KI and mCherry were used as controls. Images were taken 3 days postagroinfiltration (dpa). All experiments were performed in triplicate and showed similar results. Scale bars, 1 cm. (b) Electrolyte leakage from infiltration sites was measured as a percentage of leakage from boiled samples. Wilcoxon rank-sum test was used for statistical analysis. Error bars represent SD; asterisks indicate 0.01 < p < 0.05. ns, not significant.
Western blot analysis confirmed expression of each effector protein in the leaves ( Figure 4h). Thus, our findings suggest that both PiAvr3b isoforms enhance plant susceptibility by suppressing PAMP-and effector-associated cell death.

| Identification of residues in PiAvr3b that regulate plant defence
To dissect the PiAvr3b residues responsible for regulating plant defence, we selected PiAvr3b RGR as a test case because no functional variation was observed between the two PiAvr3b isoforms.
We made six truncation mutants of PiAvr3b RGR (DM1-DM6) ( Figure 5a) and tested their ability to activate StR3b-mediated HR and suppress cell death triggered by PiNpp or Pi10232. We found that the fragment containing residues 58-145 (DM4) was the smallest fragment that could initiate StR3b-mediated HR and suppress PiNpp-mediated necrosis. Fragments lacking residues 125-145 were only capable of suppressing Pi10232-mediated cell death (Figure 5b-d).
To further characterize the residues contributing to the dual activities of PiAvr3b, we performed a mutational analysis of PiAvr3b based on the identified sequence polymorphism (Figures 1 and 6a).
When the conserved tryptophan was mutated (W60A), PiAvr3b could not stimulate StR3b-mediated HR or suppress PiNpp-or Pi10232-induced cell death (Figure 6b-d). In comparison, alanine substitution of glutamic acid (E72A), aspartic acid (D76A), tryptophan (W77A), isoleucine (I97A), or arginine (R124A) did not affect the dual activity of PiAvr3b. Intriguingly, we found that the mutation S99A partially attenuated suppression of the PiNpp-triggered cell death but did not alter StR3b activation or Pi10232 suppression (Figure 6b,c). Next, we focused on the E134 residue, which was replaced by aspartic acid (E134D) in StR3b-inactivating orthologs. We demonstrated that the E134 residue is also important for suppressing PiNpp-and Pi10232-induced necrosis and activating The Wilcoxon rank-sum test was used for statistical analysis. Two asterisks indicate statistical difference (Student's t test, p < 0.01). These experiments were repeated three times with similar results.

F I G U R E 4
Expression of PiAvr3b isoforms in Nicotiana benthamiana enhances Phytophthora infestans infection and suppresses pathogenassociated molecular pattern (PAMP)-and effector-induced cell death. (a) Symptoms of P. infestans infecting N. benthamiana leaves that transiently expressed PiAvr3b isoforms. Pi18221 is a truncated paralog of PiAvr3b. All constructs shown were expressed as mCherry fusions. mCherry and PiAvr3a KI served as controls. Pictures were taken 5 days after P. infestans zoospore inoculation. Dotted lines outline lesions. (b) Quantification of lesion sizes such as shown in (a). Pooled data from three independent experiments that comprise eight to 10 inoculations are presented as mean ± SD. Asterisks indicate significant differences (p < 0.01) based on the Wilcoxon rank-sum test; ns, not significant. (c) Quantitative measurement of relative P. infestans biomass shown in (a). P. infestans biomass in N. benthamiana leaves expressing PiAvr3b isoforms, Pi18221, or PiAvr3a KI , was determined via quantitative PCR and normalized to the mCherry control. Kruskal-Wallis and Dunn's tests were applied for statistical analysis, and letters indicate statistically different values (p < 0.01). (d) Numbers of sporangia isolated from diseased tissues shown in (a). The y axis shows sporangia numbers (per lesion) recovered at 6 days postinoculation from each treatment. Error bars depict SD. Two asterisks indicate sporangial numbers significantly different from the mCherry control; ns, not significant (Wilcoxon rank-sum test, p < 0.01). (e) Reactive oxygen species production in N. benthamiana leaf discs overexpressing mCherry, PiAvr3b RGR , PiAvr3b LRK , Pi18221, PiAvr3a KI , or Pi20303 after treatments of 100 μM flg22 or water control. The relative luminescent units (RLU) were detected over a 45-min period. Mean values (±SD) of eight replicates are shown. This experiment was repeated three times and showed similar results. (f) Cell death responses on N. benthamiana leaves co-expressing PiAvr3b variants and Phytophthora-originated PAMPs. Agrobacterium strains harbouring PiNpp, PiINF1, PsXEG1, Pi08174, or Pi10232 were co-infiltrated with Agrobacterium strains expressing mCherry, PiAvr3b RGR , PiAvr3b LRK , Pi18221, or PiAvr3a KI at a 1:1 ratio and a final OD 600 of 0.4. Ratios on pictures indicate the agroinfiltrated areas that showed necrosis. Twelve or 18 agroinfiltration sites were photographed after 3-5 days. The experiment was repeated three times with similar results. Scale bars, 5 mm.

| Nuclear localization of PiAvr3b is critical for triggering HR and suppressing PTI, but not required for inhibiting effector-induced necrosis
Previous studies have shown that PiAvr3b localizes to both the nucleus and cytosol (Zheng et al., 2014). To examine whether subcellular localization determines the activity of PiAvr3b, we F I G U R E 5 Mutational analysis of the PiAvr3b RGR segments that contribute to StR3b-mediated hypersensitive response (HR) and PiNpp-or Pi10232-triggered cell death. (a) Schematics illustrating the six truncated PiAvr3b RGR fragments (DM1-DM6) tested in the cell death assay. The dashed rectangle denotes the signal peptide (Signal P). The mature PiAvr3b RGR (without signal P) was used as a wildtype (WT) control. Green bars represent the predicted α-helices at the C-terminus of PiAvr3b using Jpred4 program (http://www.compb io.dundee.ac.uk/jpred/). Fragments that stimulated StR3b-mediated HR and suppressed PiNpp-and Pi10232-induced necrosis are coloured purple. PiAvr3b RGR truncations that retained only the ability to suppress Pi10232-induced cell death are coloured blue. Fragments coloured grey represent nonfunctional mutants. (b) Functional analysis of the mCherry-tagged PiAvr3b RGR truncations shown in (a) on Nicotiana benthamiana leaves expressing StR3b, PiNpp, or Pi10232. Pictures were taken 3-5 days postagroinfiltration. Ratios indicate the fraction of agroinfiltrated leaf areas that developed necrosis. Eighteen agroinfiltration sites were tested in each experiment; two independent experiments showed similar results. Scale bars, 5 mm. (c) Quantification of cell death in (b) by measuring electrolyte leakage. Electrolyte leakage from infiltration sites was measured as a percentage of leakage from boiled samples. Error bars represent the SD. Different letters at the tops of bars represent significant differences (Kruskal-Wallis and Dunn's tests, p < 0.05.). (d) Incubation with α-mCherry shows fusion proteins expressed in N. benthamiana. Protein loading was indicated by Ponceau S staining. altered protein localization by fusing a nuclear localization signal (NLS) and an endoplasmic reticulum (ER) retention signal (signal peptide + HDEL) to the mCherry-fused PiAvr3b RGR generated previously, which formed PiAvr3b RGR -NLS and PiAvr3b RGR -HDEL. Employing high-resolution confocal microscopy, we found that PiAvr3b RGR -NLS predominantly localized in the nucleus, while PiAvr3b RGR -HDEL accumulated in the ER and cytosol ( Figures S7 and 7a). Relative to PiAvr3b RGR and PiAvr3b RGR -NLS, little fluorescent signal of PiAvr3b RGR -HDEL was observed in the nucleus (Figure 7a). This observation was confirmed by colocalizing PiAvr3b RGR -HDEL with ER markers, but not nuclear markers ( Figure S8). In parallel, we tested the activity of these mislocalized proteins in cell death suppression and StR3b activation. As shown in Figure 7b,c, similar to PiAvr3b RGR , PiAvr3b RGR -NLS triggered StR3b-mediated HR and suppressed PiNpp-induced cell death, whereas PiAvr3b RGR -HDEL lost both activities. This implies that nuclear accumulation is required for PTI suppression and ETI induction of PiAvr3b RGR . Surprisingly, we found that Avr3b RGR -NLS could not inhibit cell death induced by the RxLR effector Pi10232, while the ER-localized PiAvr3b RGR -HDEL remained capable of suppressing Pi10232-induced necrosis (Figure 7b,c).
Western blot analysis confirmed expression of these constructs F I G U R E 6 Mutational analysis of PiAvr3b RGR residues involved in StR3b-mediated activation and immune suppression. (a) The amino acid sequence of PiAvr3b RGR . The RxLR-EER motif is highlighted in red. Black arrows and corresponding numbers indicate residues subject to alanine scanning. (b) Nicotiana benthamiana leaves showing cell death triggered by StR3b, PiNpp, or Pi10232 and alanine substitution mutants of PiAvr3b (M1-M8). mCherry-PiAvr3b RGR (WT) was used as a control. Pictures were taken 3-5 days postagroinfiltration. Ratios indicate the fraction of agroinfiltrated leaf areas that developed necrosis. Eighteen agroinfiltration sites were included in each experiment; two independent experiments were performed and displayed similar results. Scale bars, 5 mm. (c) Quantification of cell death in (b) by measuring electrolyte leakage. Electrolyte leakage from infiltration sites was measured as a percentage of leakage from boiled samples. Error bars represent SD. Kruskal-Wallis and Dunn's tests were applied for statistical analysis with a p-value cutoff <0.05. Different letters at the top of each bar represent significant differences. Two independent biological replicates were performed with similar results. (d) Immunoblot analysis of expressed fusion proteins using α-mCherry. Protein loading was indicated by Ponceau S staining.
in N. benthamiana (Figure 7d). These results imply that nuclear localization of PiAvr3b is indispensable for HR induction and PTI suppression activities, although it is not required for ETI suppression ( Figure 7e).

| DISCUSS ION
In this study, we demonstrated genetic and transcriptional variation among PiAVR3b alleles in 95 naturally isolated P. infestans strains.
PiAVR3b amino acid polymorphisms grouped the effector into two isoforms. We found that PiAvr3b protein sequences were only conserved in Phytophthora Clade I, and not all orthologs were capable of triggering cell death in the presence of the R protein StR3b. We found that HSP90 and SGT1 were required for PiAvr3b-inducing cell death, and both PiAvr3b isoforms promoted P. infestans infection by suppressing different cell death inducers. Based on the mutational analysis, we identified distinct regions of PiAvr3b that determine its activity in cell death activation and plant immunity suppression.
We also further illustrated that nuclear localization of PiAvr3b was critical for its HR elicitation and PTI inhibition activities, but not for suppression of effector-induced necrosis.
Our results showed that gene deletion and silencing of PiAvr3b are responsible for evasion of StR3b-mediated HR, which possibly contributed to the rise of super-virulent P. infestans strains that overcame the R1-R11 resistance genes in potatoes (Cooke et al., 2012;Fry, 2008;Tian et al., 2015). We found that PiAvr3b was only conserved across the six P. infestans sister species in the Phytophthora taxonomy Clade I, and not all of the PiAvr3b orthologs in these species could induce StR3b-mediated cell death. Interestingly, other RxLR effectors with low sequence similarity, such as P. sojae Psoj292825 and P. infestans Pi19831, could trigger HR. This finding supports the view that effectors recognized by NLRs are likely to be functionally conserved and thus may be exploited in developing F I G U R E 7 Mis-targeting of PiAvr3b alters StR3b-induced hypersensitive response (HR) and PiNpp-or Pi10232-triggered cell death. (a) Subcellular localization of PiAvr3b RGR fused with (mis)targeting signals in Nicotiana benthamiana. These include a nuclear localization signal (NLS) and an endoplasmic reticulum (ER) retention signal (HDEL), and mCherry-PiAvr3b RGR (WT) was used as a control. Images were captured at 2-3 days postagroinfiltration (dpa). Scale bars, 10 μm. The fluorescence density across the nucleus (grey bar) was measured and compared, as shown on the right panel. Three independent confocal microscopy observations (n > 18) were made, and representative images are shown. broad-spectrum resistance (Lin et al., 2022). Based on the guard hypothesis, multiple effectors that target the same host protein do not need to be conserved in sequence to be recognized by the R protein guarding that target (Marathe & Dinesh-Kumar, 2003). The presence of multiple PiAvr3b variants in diverse Phytophthora pathogens suggests that solanaceous crops maintain an active StR3b-specific immune system, thus providing durable resistance to late blight disease. Multiple NLR genes, including StR3b in the wild potato (S. pinnatisectum), confer resistance to numerous prevalent races of P. infestans (Gu et al., 2020), suggesting that StR3b and its functional equivalents remain a useful resistance gene to control Phytophthora infection.
The broad recognition spectrum of StR3b illustrates the high level of complexity in R genes associated with the plant's immune surveillance system. In N. benthamiana, we found that the key ETI signalling components HSP90 and SGT1 were required for PiAvr3b-StR3b-induced HR. A similar observation was made for the interaction between R3a and PiAvr3a KI , in which both SGT1 and HSP90 are involved in the ETI response (Bos et al., 2006).
Interestingly, HSP90 and SGT1 have been documented as essential regulators for INF1-and NIP-induced cell death , suggesting crosstalk between the PRR and NLR signalling pathways. In addition, PiAvr3b suppressed cell death triggered by the RxLR effector Pi22798, and its necrosis-inducing activity also relied on SGT1 . In Arabidopsis and N. benthamiana, SGT1, HSP90, and RAR1 form chaperone complexes to regulate ETI (Shirasu, 2009), but our results showed that RAR1 was not necessary for StR3b-mediated cell death. Mitogenactivated protein kinases (MAPKs), such as MAPKKK and MEK2, are associated with PTI and ETI (Pedley & Martin, 2005). Silencing of MEK2 compromised the RxLR effector PsAvh238-induced cell death in N. benthamiana (Yang et al., 2017). However, this protein was dispensable for StR3b-associated HR, indicating that HR induced by the StR3b-PiAvr3b interaction may have a different downstream signalling pathway.
Extensive studies on Phytophthora effectors suggest that most RxLR effectors could enhance P. infestans colonization (Wang et al., 2019), and their activities are often associated with PTI suppression (Yin et al., 2017). PiAvr3b was previously reported to suppress flg22-induced PTI and PiNpp-triggered cell death in tomatoes (Zheng et al., 2014;Zuluaga et al., 2016). Conversely, we found that both PiAvr3b RGR and PiAvr3a KI failed to interrupt the flg22-induced ROS burst in N. benthamiana. The discrepancy between these results may be due to differences in PTI components among diverse plants.
Our results showed that PiAvr3b strongly suppressed the immune responses triggered by different PAMPs, such as PiINF1, PsXEG1, and PiNpp. As distinct plant immune receptors recognize these PAMPs, PiAvr3b probably inhibits a shared signalling pathway associated with plant immunity. The MAPK pathway is worthy of further examination because PiAvr3b suppressed flg22-dependent posttranslational MAP kinase activation in tomatoes (Zheng et al., 2014).
Moreover, we noticed that PiAvr3b suppressed cell death triggered by the effector Pi10232 but not Pi08174, while an opposite observation was made for PiAvr3a KI (Figure 4f). This difference may be due to distinct signalling pathways targeted by PiAvr3a KI and PiAvr3b (Whisson et al., 2016). To better understand the mode of action of PiAvr3b, further investigation may be needed to identify proteins that directly interact with PiAvr3b.
The integrity of the effector protein functional domain is critical for bioactivity. We found that Pi18221, the truncated paralog of Avr3b, could not elicit StR3b-associated cell death (Figure 2a,b).
This was consistent with our mutational assay, in which deletion of the PiAvr3b C-terminal domain (residues 58-145) impaired StR3b activation. Our attempt to find a single residue that could separate the dual activities of PiAvr3b was unsuccessful. This suggests that the HR-induction and necrosis-suppression activities are linked, even though mutation of S99A in PiAvr3b partially compromised suppression of PiNpp-associated necrosis. The same effector domain controlled the suppression of PiNpp-induced cell death and activation of StR3b, indicating possible structural similarity between the potential interacting targets. Therefore, deletion or silencing of PiAVR3b could be the most effective way for P. infestans to escape host surveillance during pathogen-host coevolution.
A large portion of the RxLR effectors tested thus far were localized in plant nuclei or cytoplasm (Wang et al., 2019). However, the relationship between subcellular dynamics and the bioactivity of various RxLR effectors is less well illustrated. Studies have shown that nucleocytoplasmic transport of RxLR effectors PiAvr1 and PsAvh238 is required for PTI suppression or cell death induction (Du et al., 2015;Yang et al., 2017). Relocalization of PiAvr3a KI from the cytosol to endosomes was associated with activation of the corresponding R gene during infection (Engelhardt et al., 2012). Transient expression assays demonstrated that PiAvr3b was distributed in both the N. benthamiana cytosol and nucleus (Zheng et al., 2014), and was not altered during infection. To clarify the correlation between the subcellular localization and bioactivities of PiAvr3b, we attempted to force PiAvr3b to accumulate solely in the cytoplasm by adding a nuclear export signal (NES) (Figures S9-S11). However, we observed that PiAvr3b-NES could still sufficiently trigger StR3b-HR and suppress PiNpp-necrosis ( Figure S9). This result differs from a previous report that showed that NES-tagged PiAvr3b was nonfunctional (Wang et al., 2022). One possible explanation for this difference could be that a single NES is insufficient to export all of the protein from the nucleus, and in fact we observed c.25% fluorescent signal that still resided in the nuclei ( Figure S9a). Alternatively, we tried to exclude PiAvr3b from the nucleus by adding an ER retention signal (HDEL), and PiAvr3b RGR -HDEL no longer activated ETI, suggesting that nuclear localization is required for StR3b recognition.
Consistent with these findings, balanced nucleocytoplasmic transportation of the P. infestans effector Avr1 was reported to regulate the activation of R1-mediated HR and suppression of CRN2-induced cell death (Du et al., 2015).
Because of the sequence similarity between StR3a and StR3b, StR3b was predicted to accumulate in the host cytoplasm and has a resistance mechanism similar to R3a (Li et al., 2011). Our data provide new evidence supporting a mechanistic distinction between these two R proteins. Therefore, it is crucial to identify the host targets that mediate interactions between StR3b and PiAvr3b to understand how this resistance protein can recognize a variety of RxLR effectors. Because the same amino acid substitution compromises both StR3b-activation and PiNpp-suppression, it is logical to envision a model that involves interactions with a plant target protein guarded by StR3b. It also seems plausible that Pi19831 and other PiAvr3b variants bind to the plant target protein and thereby trigger R3b-dependent HR. The well-known PTI-negative regulator RIN4 can be modified by many different bacterial Avr proteins and is, therefore, guarded by the resistance proteins RPM1 and RPS2 (Jones & Dangl, 2006). Our study may represent another example of a potential host protein associated with a filamentous plant pathogen effector and its corresponding NB-LRR immune receptor.

| Phylogenetic analysis
All PiAvr3b orthologs were identified by reciprocal BLAST searches.
Alignment of the mature PiAvr3b protein sequences and its homologs was implemented by MEGA X software (v. 10.0.5) (Kumar et al., 2018). The phylogenetic neighbour-joining (NJ) tree was constructed using the Poisson model with default settings (uniform rates, pairwise deletion, and 1000 bootstraps).

| P. infestans culture and plant growth conditions
All P. infestans isolates used in this study (Table S1) were maintained on rye-sucrose agar at 18°C in the dark. Zoospores were prepared according to the method by Yin et al. (2017). The concentration of zoospores was adjusted to approximately 100/μl before inoculation.

| Plasmid construction
The plasmids and primers used in the study are listed in Tables Table S4. NLS and NES/nes were used as previously reported (Du et al., 2015;Yang et al., 2017). To generate the ER-targeting mutants, the signal peptide of N. benthamiana endochitinase (SOL ID: Niben101Scf07491Ctg009) and the ER retention signal (HDEL) were PCR amplified and fused to mCherry-PiAvr3b.

| HR and cell death suppression tests
Agrobacterium tumefaciens GV3101 was used for the microinfiltration, and the plasmids harbouring desired genes were introduced into A. tumefaciens via the freeze-thaw method (Weigel & Glazebrook, 2006). Microinfiltration was carried out as follows.

| VIGS assays in N. benthamiana
pTRV (Liu et al., 2002) was used as the backbone plasmid for all the VIGS constructs, including pTRV1, pTRV2-GFP, pTRV2-HSP90, pTRV2-SGT1, pTRV2-MEK2, and pTRV2-RAR1. A. tumefaciens GV3101 was used and adjusted to an OD 600 of 0.8 for the agroinfiltration. pTRV1 and pTRV2 constructs were co-infiltrated into the two primary leaves of a plant at the four-leaf stage. The efficacy of gene silencing was examined via reverse transcription-quantitative PCR (RT-qPCR) using Nbactin (Table S4) as the reference gene.

| Immunoblot analysis of transiently expressed proteins
Agroinfiltration of mCherry-tagged RxLR effectors was conducted using 4-to 6-week-old N. benthamiana leaves. Forty-eight to 72 h after agroinfiltration, 1-cm 2 leaf discs were excised and ground into fine powders in liquid nitrogen. Five hundred microlitres of lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP40, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA) supplemented with 1% protease inhibitor cocktail was added to each pulverized plant sample. Lysates were centrifuged at 4°C, 18,759 × g for 10 min, and supernatants were transferred to fresh tubes. For each sample, 100 μl of supernatant was mixed with 100 μl of 2× loading buffer, heated at 95°C for 5 min, and kept on ice. Ten microlitres of the protein samples was separated via 10% SDS-PAGE and transferred to a 0.45μm nitrocellulose membrane (Cytiva) at 10 V for 1-2 h. Skimmed milk 5% (wt/vol) was used to block the membrane. Mouse monoclonal mCherry antibody (Abcam) was used as the primary antibody at 1:10,000 dilution. The membrane was washed with phosphatebuffered saline with 0.1% Tween 20 before adding the secondary goat anti-mouse immunoglobulin (IgG) horseradish peroxidase conjugate (Abcam) at 1:2000 dilution. The SuperSignal West Femto substrate (Thermo Scientific) for protein detection was used according to the manufacturer's instructions.

| RT-qPCR
Total RNA was isolated from infected N. benthamiana leaves with the RNeasy Plant Mini Kit (Qiagen). Total RNA (1 μg) was used for reverse transcription. First-strand cDNA was synthesized using SuperScript III (Thermo Fisher) and qPCR was conducted employing the SYBR Premix Ex Taq II kit (Takara) according to the manufacturer's instructions.

| P. infestans inoculation, biomass assay, and sporangial measurement
To evaluate the gene's function, N. benthamiana leaves were detached and transferred to a Petri dish 24 h after agroinfiltration.
Infiltrated regions were inoculated with 10 μl of P. infestans Pi009 zoospores suspension (10 5 zoospores/ml) and incubated at 25°C in the dark as previously described (Gu et al., 2020). Plants were photographed under high-intensity UV light (Analytic Jena) at 5 days postinoculation. Infected areas on the leaves were quantified by ImageJ. Three independent biological replicates were included.
To perform biomass measurement, total gDNA was extracted using a hexadecyltrimethylammonium bromide method (May & Ristaino, 2004) and quantified by 1% agarose gel electrophoresis. Quantitative PCR of gDNA was performed using CFX Connect (BioRad) as described by Gu et al. (2021) to obtain biomass measurements. Genomic DNA abundance or relative gene expression was measured by a 2 −ΔΔCt method using the Actin genes from both P. infestans and N. benthamiana as endogenous controls. To count sporangial numbers, whole leaves displaying lesions were immersed in 3 ml of water and vortexed vigorously. The number of sporangia harvested from each leaf was counted using a light microscope (BX63; Olympus).

| Measurement of ROS
ROS production was examined using a luminol/peroxidase-based method (Albert et al., 2015). Briefly, 0.4 cm leaf discs were collected from 6-week-old N. benthamiana leaves and floated on 200 μl of sterile water (using 96-well plates) overnight. The following day, water was replaced with the luminol (35.4 μg/ml)/peroxidase (10 μg/ml) reaction solution (dissolved in sterile water), and 100 nM of the flagellin-derived peptide flg22 (Genscript Biotech Corp.) or an empty vector. Luminescence was measured by the Varioskan LUX microplate reader (Thermo Fisher Scientific).

| Electrolyte leakage assay
An electrolyte leakage assay was conducted to evaluate PAMPor effector-triggered cell death, according to a previously described method (Yang et al., 2017). Briefly, for each sample, five leaf discs (1 cm in diameter) from agroinfiltrated areas were collected and floated on 5 ml of distilled water overnight at 25°C. The next day, the conductivity of the bathing solution was measured using a conductivity meter (FE32 FiveEasy; Mettler Toledo), which yielded value A. The leaf discs in the bathing solutions were boiled in sealed tubes for 30 min. After cooling the solution to 25°C, the conductivity was measured again to obtain value B. Ion leakage was measured as a percentage of total ions: (value A/value B) × 100. In the case of agroinfiltration, the ratio of total ions was compared using the Wilcoxon rank-sum test. Multiple independent measurements were compared by Kruskal-Wallis and Dunn's multiple comparisons tests.

| Confocal microscopy
The laser scanning confocal microscope LSM 900 with Airyscan 2 (Zeiss) was used to examine the expression and subcellular localization of GFP-or mCherry-fused proteins in N. benthamiana leaves. Images were captured using a 40× water objective lens with excitation/emission settings of 561 nm/570-630 nm for mCherry and 488 nm/495-555 nm for GFP under super-resolution. Single optical section images were acquired from leaf epidermal cells, and z-stacks were collected from leaf cells and projected and processed using the Carl Zeiss ZEN blue 3.2.
Images were edited by Adobe Photoshop CS5 v. 12.0.1.

| Yeast two-hybrid assay
The yeast two-hybrid assay was conducted using the Matchmaker system (Clontech). Briefly, the bait plasmid pGBKT7-PiAvr3b RGR and the prey plasmid pGADT7-StR3b were introduced into Saccharomyces cerevisiae AH109, and nutritional selection (triple dropout [TDO]/−Trp− Leu−His and quadruple dropout [QDO]/−Trp−Leu−His−Ade) was used to recover transformants. The pGBKT7-53 and pGADT7-T plasmid pair was used as a positive control, and the empty plasmids pGBKT7 and pGADT7 were applied to rule out self-activation of the reporter genes.

ACK N O WLE D G E M ENTS
We are grateful to Guangcun Li (Institute of Vegetables

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.
[Correction added on 20 February 2023, after first online publication: